Application of METTL3 inhibitor in preparation of medicine for inhibiting PI3K/Akt and ERK1/2 signal channels

A technology of signaling pathways and inhibitors, applied in drug combinations, antineoplastic drugs, pharmaceutical formulations, etc., can solve the side effects of inhibitors and other problems, and achieve the effect of promoting progress

Pending Publication Date: 2022-03-15
HARBIN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the problem of side effects of existing PI3K/Akt and ERK1/2 signaling pathway inhibitors, and p...

Method used

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  • Application of METTL3 inhibitor in preparation of medicine for inhibiting PI3K/Akt and ERK1/2 signal channels
  • Application of METTL3 inhibitor in preparation of medicine for inhibiting PI3K/Akt and ERK1/2 signal channels
  • Application of METTL3 inhibitor in preparation of medicine for inhibiting PI3K/Akt and ERK1/2 signal channels

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Establishment of METTL3 knockdown HCT116 cell line

[0035] Firstly, a guide RNA (sgRNA) for frameshift mutation of METTL3 was designed using Crispr Can (https: / / www.Crispr.org) and Crispr Direct (http: / / Crispr.dbcls.jp) websites, and the sequence is GAGAGGCTGCAGCGGAGG. The double-stranded DNA was annealed, and inserted into the PX458 vector after digestion, ligation and transformation to obtain a knockdown METTL3 vector. Then use the transfection reagent Lipofectamine3000 to transfer the above recombinant plasmids into HCT116 cells. After puromycin selection, they were separated into 96-well plates by limiting dilution method, and single clones were selected to detect the knockdown efficiency by qRT and Western Blot. The METTL3 knockdown HCT116 cell line was obtained.

Embodiment 2

[0036] Example 2: Effects of METTL3 on proliferation, invasion, migration and clone formation of intestinal cancer cells

[0037] 1. The effect of METTL3 on cell proliferation

[0038] First, the wild-type and METTL3 knockdown HCT116 cells established in Example 1 were inoculated in 6-well plates, and after 12 hours of attachment, the Vector plasmid (GV230 overexpression empty vector) was transferred into wild-type HCT116 using lipo3000 transfection reagent In the 6-well plate of cells, vector plasmid (GV230 overexpression empty vector), GV230-EphA2 plasmid (overexpression EphA2), GV230-VEGFA plasmid (overexpression VEGFA) and two EphA2 and VEGFA overexpression plasmid was transformed into METTL3-deficient HCT116 cells. After 48 hours, EphA2 and VEGFA were successfully restored by qRT-PCR and western blot analysis. At the same time, collect the cells in the 6-well plate after 48 hours of lipo3000 transfection, and add 1×10 3 Cells were seeded in 96-well plates, filled with ...

Embodiment 3

[0048] Example 3: Effect of METTL3 on Vascular Mimicry

[0049] 1. Detection of EphA2 and VEGFA in HCT116 cells

[0050] The wild-type and METTL3-knockdown cells obtained in Example 1 were cultured to the third passage, recovered cells, added RIPA lysate, sonicated at 300W for 8-10s, placed on ice for 10min, centrifuged at 10000r / min at 4°C for 10min, and aspirate the supernatant Take 2 μL to measure the protein concentration with the BCA protein concentration assay kit, add the protein concentration to the loading buffer, and centrifuge in a boiling water bath for 5 minutes. Put the prepared protein gel into the electrophoresis tank and add an appropriate amount of electrophoresis buffer. After electrophoresis with the stacking gel at 80V and the separating gel at 120V, turn off the power, take out the electrophoresis clip, and carefully rinse the foam on the film with tap water. Take a clean culture dish, pour an appropriate amount of transfer solution, and transfer the cut...

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Abstract

The invention discloses application of an METTL3 inhibitor in preparation of a medicine for inhibiting PI3K/Akt and ERK1/2 signal channels, belongs to the technical field of intracellular signal channels, and aims to solve the problem that the existing PI3K/Akt and ERK1/2 signal channel inhibitors have side effects. The invention relates to an application of an METTL3 inhibitor in preparation of drugs for inhibiting PI3K/Akt and ERK1/2 signal pathways. Knock-down of METTL3 can inhibit proliferation of colon cancer cells, invasion, migration and clone formation of colon cancer cells, expression of EphA2 and VEGFA, and formation of vascular mimicry. The method is applied to the field of screening of colon cancer targeted drugs.

Description

technical field [0001] The invention belongs to the technical field of intracellular signaling pathways, and specifically relates to an application of METTL3 in inhibiting PI3K / Akt and ERK1 / 2 signaling pathways. Background technique [0002] Colorectal cancer is the third most common cancer and the second leading cause of cancer-related death in the world. Although new diagnostic techniques and treatment methods are updated day by day, the morbidity and mortality of CRC patients are still high. Epigenetics has been shown to play a crucial role in the initiation and progression of cancer. N6-methyladenosine (m6A) is the most common modification on mRNA in most eukaryotes. The m6A modification mainly occurs in the adenine of the "RRACH" motif, and the process is catalyzed by the "writer" (METTL3, METTL14, WTAP, etc.), and the methylation that occurs can be reversed by the "eraser" (ALKBH5, FTO). Proteins that recognize and bind to m6A methylation sites on RNA are called "re...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/7105A61P35/00A61P35/04
CPCA61K45/00A61K31/7105A61P35/00A61P35/04
Inventor 吴琼贺洪娟刘鑫滕祥琦李佳琪李博然
Owner HARBIN INST OF TECH
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